Barium 4-dodecylphenolate

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Remarks:

Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-08-05 to 2020-12-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-08-05 to 2020-12-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed 2019-09-16

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (21 to 29°C), in a dark and dry room in the original container

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:SD

Details on species / strain selection:

Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Bioneeds India Private Limited (in-house bred animals)
- Females nulliparous and non-pregnant: yes

- Age at study initiation (treatment day 1):
Males: 87 to 95 days (12 to 13 weeks)
Females: 87 to 95 days (12 to 13 weeks)

- Weight at study initiation (treatment day 1):
Males: 271.93 to 326.31 g (main and recovery groups)
Females: 222.79 to 247.13 g (main and recovery groups)

- Housing: housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube; bedding material: clean sterilized paddy husk
- Acclimatization: maximum of three animals/sex were housed together
Main group animals:
- Pre-mating: 2 animals/sex/group were housed together
- Cohabitation Period (mating) - 1 male / 1 female of the same group were housed together
- Post-mating: males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as nesting material for main group females from gestation day 20 onwards. Lactating females were housed individually with their offspring.
Recovery Group Animals: maximum of three animals/cage per group of the same sex were housed.

- Diet (ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (ad libitum): deep bore-well water passed through reverse osmosis unit

- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19.8 to 23.2 °C
- Relative humidity: 49 to 64 %
- Air changes: 12 to 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Details on route of administration:

The vehicle and test item formulations were administered through oral (gavage) route as it is a possible route of exposure to human.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulations were freshly prepared before dose administration on each treatment day and they were administered as soon as possible after preparation.

The required quantity of test item was weighed into a clean glass beaker. A little volume of vehicle was added into the beaker and mixed well with glass rod and then placed on a hot plate magnetic stirrer with a set temperature of 40 to 60°C. The formulation was allowed to stir under this condition by adding little quantity of vehicle to facilitate homogenous sampling. After confirmation of uniform suspension of formulation, the entire content was transferred into the measuring cylinder and the beaker was rinsed using a small volume of vehicle and the volume was transferred to measuring cylinder. The rinsing procedure was repeated until entire quantity of the test item formulation was transferred into a measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get a desired concentration of test item. After that, the entire quantity was transferred to beaker, allow for stirring by placing on a hot plate magnetic stirrer with a set temperature of 40 to 60°C until uniform suspension of test item formulation was obtained.

The test item formulations were allowed to cool down at room temperature prior to administration. The test item formulations were maintained under stirring conditions using magnetic stirrer to maintain homogeneity of the test item formulations during test item administration.

Administration volume: 4 mL/kg bw
The actual dose volume for each animal was calculated based on the most recent body weight.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
The test item was not soluble in distilled water and could not be suspended in
0.5 % w/v carboxy methyl cellulose at the concentration of 25 mg/mL (the highest dose concentration considering an equivolume of 10 mL/kg body weight). The test item formed a uniform suspension with corn oil at the concentration of 125 mg/mL (the highest dose concentration considering equivolume of 4 mL/kg body weight) as per in-house miscibility test results.
Corn oil was used as vehicle for test item formulations in the range finding study. For consistency, this is the preferred vehicle for the present study.
Corn oil is universally accepted and routinely used vehicle in oral toxicity studies. Hence, corn oil was selected as vehicle for test item formulations.

- Manufacturer: MP Biomedicals
- Lot/batch no.: L32011001
- Expiry date: February 2023

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of test item in dose formulations were established. The test item barium 4-dodecylphenolate was stable for 48 hours at room temperature in corn oil at the concentrations of 5 mg/mL and 150 mg/mL.

In addition, homogeneity and dose formulation analysis for dose concentration verification was conducted. For the determination of the test item concentration in the prepared test item formulations, samples were taken and stored at -20°C ± 10% until analysis. Sampling and analysis of formulations was performed during week 1, week 4 and week 7 of the treatment. The samples were collected in duplicates from top, middle and bottom layers from control (vehicle), low (50 mg/kg bw/day), low intermediate (100 mg/kg bw/day), high intermediate (200 mg/kg bw/day) and high dose concentrations (450 mg/kg bw/day) and in duplicates from single layer from vehicle control.

One set of aliquots of each formulation was analyzed. The second aliquot was stored as a backup purpose at established stability conditions. Formulations were considered acceptable, since the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is less than 10%.

The following ranges were determined for the treatment groups:
Recovery of formulations (%).
50 mg/kg bw/day: 93.18 % - 100.15 % (% RSD: 0.41 - 2.44)
100 mg/kg bw/day: 92.93 % - 96.09 % (% RSD: 0.60 - 3.18)
200 mg/kg bw/day: 93.80 % - 107.04 % (% RSD: 0.30 - 1.62)
450 mg/kg bw/day: 94.46 % - 106. 47 % (% RSD: 0.53 - 0.84)

Duration of treatment / exposure:

1) Main group:
- males: two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 46 days of treatment)
- females (pregnant/littered): two-week pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 (ranging from a total period of 49 to 67 days)
-females (mated but non-pregnant): two-week pre-mating period, during mating until confirmed as mated and further 24 days from the day of confirmation of mating (ranging from a total period of 43 to 57 days)
- females: (without positive mating signal): two-week pre-mating period, during the entire mating period i.e. 21 days and further 24 days from the day of termination of mating (with a total period of 59 days)

2) Recovery group:
- males and females: 52 days

Frequency of treatment:

once daily, 7 days/week

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

Main group

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Main group

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

Main group and recovery group

Dose / conc.:

450 mg/kg bw/day (actual dose received)

Remarks:

Main group and recovery group

No. of animals per sex per dose:

Main groups: 12 males / 12 females
Recovery groups: 5 males / 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: for dose selection, information from an acute oral toxicity study (please refer to IUCLID section 7.2.1 Acute toxicity: oral: BaDodec_k_Niklas_2018) with barium 4-dodecylphenolate was used, since substance-specific data are limited and this test is the most valuable with regard to systemic toxicity. In this study, the No Observed Adverse Effect Level [NOAEL] of test item barium 4-dodecylphenolate is 300 mg/kg body weight. Hence, the doses of 0, 20, 100, 250 and 500 mg/kg body weight/day were selected as control, low, low mid, high mid and high dose groups for a dose range finding study performed at the laboratory. Groups of 5 male and 5 female Sprague Dawley rats were administered via gavage with the test item in corn oil at the four dose levels. A vehicle control group was run concurrently.

Results:
There were no treatment related significant changes in any of the systemic parameters in either sex noted at 20, 100 and 250 mg/kg body weight/day. However, significant reduction in body weight, food consumption and reproductive organ weights (prostate along with seminal vesicles with coagulating gland) were noted at 500 mg/kg body weight/day in males only in the dose range finding study.

Based on the results obtained from dose range finding study, the doses of 0, 100, 200, 450 and 50 mg/kg body weight were selected for the present study.

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked: clinical signs and mortality/morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: day 1 before treatment and weekly thereafter during treatment

BODY WEIGHT: Yes
- Time schedule for examinations:
Main group males: at receipt, on the day of randomization, on the first day of dosing, weekly thereafter and at termination

Main group females: at receipt, on the day of randomization, on the first day of dosing, weekly thereafter during pre-mating and until confirmation of mating.

Main group females (pregnant): gestation days 0, 7, 14 and 20 and on lactation days 1, 4, 7 and 13

Main group females (non-pregnant with and without positive mating signal): once in a week.

All the females were weighed terminally on the day of sacrifice.

Recovery group animals (males and females): at receipt, on the day of randomization, on the first day of dosing, weekly thereafter and at termination.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Main group males: cage wise food consumption was measured once a week during pre-mating period (week 1 and 2) and post-mating period (week 6).

Main group females: cage wise food consumption was measured once in a week during pre-mating period coinciding with body weight recording.

Main group females (pregnant): cage wise food consumption was measured during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4, 4 to 7 and 7 to 13.

Main group females (non-pregnant with and without positive mating signal): cage wise food consumption was measured once in a week coinciding with body weight recording during the post mating period.

Food consumption was not measured for both sexes during mating period, for the pregnant females after gestation day 20 until parturition

Recovery group animals: once per week throughout the experimental period.

Average food intake per rat (g/rat/day) was calculated using the amount of food offered and left over in each cage and the number of rats per cage. Food spillage was taken into consideration.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once before treatment for all animals, at the end of the dosing period for males (shortly prior to scheduled sacrifice, i.e. day 44), during the lactation period for pregnant females (shortly prior to scheduled sacrifice, i.e. on lactation day 13) and one day prior to sacrifice for non-pregnant females (i.e. experimental day 44 to 58) of all main group animals and during the last week (i.e. day 66) for the recovery group animals
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled sacrifice (for main group males after completion of 46 days of treatment, for main group females on lactation day 14 and for recovery group animals on scheduled sacrifice (study day 67)
- Animals fasted: Yes, overnight (water was provided ad libitum)
- Anaesthetic used for blood collection: Yes, isoflurane anesthesia
- How many animals: 5 animals/sex (main groups and recovery groups)
- Parameters checked: haemoglobin concentration, haematocrit, erythrocyte count, total leukocyte count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, mean platelet volume, differential leucocytes count, reticulocyte count, absolute reticulocyte count, absolute differential leucocytes count, prothrombin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled sacrifice (for main group males after completion of 46 days of treatment, for main group females on lactation day 14 and for recovery group animals on scheduled sacrifice (study day 67)
- Animals fasted: Yes, overnight (water was provided ad libitum)
- Anaesthetic used for blood collection: Yes, isoflurane anesthesia
- How many animals: 5 animals/sex (main groups and recovery groups)
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, cholinesterase, total protein, albumin, total bilirubin, glucose, total cholesterol, creatinine, urea, blood urea nitrogen, triglycerides, phosphate, calcium, globulin (difference between total protein and albumin value-instrument calculated), albumin/ globulin ratio, bile acids, sodium, potassium, chloride,

URINALYSIS: Yes (males only)
- Time schedule for collection of urine: at termination
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water was provided ad libitum)
- How many animals: 5 males (main groups and recovery groups) and 5 females (recovery group only)
- Parameters checked: overnight urine volume, volume, appearance, colour, blood, bilirubin, urobilinogen, ketones, protein, glucose, microalbumin, leucocytes, pH, nitrite, specific gravity and urine sediments

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Main groups: end of the dosing period for males (shortly prior to scheduled sacrifice, i.e. on day 45) and during the last week of lactation period for females (shortly prior to scheduled sacrifice, i.e. on lactation day 13)
Recovery groups: towards the end of the recovery period (shortly prior to scheduled sacrifice, i.e. on day 65)

- Dose groups that were examined: all dose groups (five animals/sex/dose group)

- Battery of functions tested:
- sensory activity: startle response, touch response, pupil response, response to nociceptive stimuli and righting reflexes were recorded.
- grip strength: hind limb and fore limb grip strength were recorded using a grip strength meter
- motor activity: motor activity was recorded using a Photoactometer/Locomotor activity monitoring system
- home cage observation: convulsions, tremors and palpebral closure and the scores were recorded
- handling observations: during removal from the cage, the animals were observed for its ease to be removed out of the cage. During handling observation, the animals were observed for the resistance, lacrimation, red and crusty deposits around eyes, nose and mouth.
- open field observations: mobility, gait, arousal, rearing, urination, defecation, stereotypes and excessive grooming in an open field arena were recorded
- neuromuscular observations: animals were dropped onto a recording paper sheet from a height of approx. 30 cm with painted paws to record the hind limb foot splay.
- physiological observation: rectal temperature was recorded using a calibrated digital thermometer

IMMUNOLOGY: No

Sacrifice and pathology:

SACRIFICE
- males from main groups were sacrificed on experimental day 47;
- pregnant/littered females from main groups were sacrificed on lactation day 14;
- mated females without positive mating signal were sacrificed after 24 days from the day of termination of mating (four females from 450 mg/kg bw/day (main group) on experimental day 60).
- mated but non-pregnant females were sacrificed after 24 days from the last day of confirmation of mating (vehicle control (main group) - one female on experimental day 44; 100 mg/kg bw/day (main group) - one female on experimental day 49; 200 mg/kg bw/day (main group) - two females on experimental days 45 and 47 each; 450 mg/kg bw/day (main group) - three females on experimental days 47, 52 and 58 each; 50 mg/kg bw/day - one female on experimental day 44).
- all males and females from recovery groups were sacrificed on experimental
day 67.

All animals were fasted overnight, water was provided ad libitum during fasting. The next day, the body weight of all fasted animals were recorded prior to exsanguination. The vaginal smear was analyzed and the stage of oestrus cycle was recorded for all main group females on the day of necropsy [i.e. lactation day 14 for littered females, 24 days after the last day of mating for mated but non-pregnant females and on experimental day 60 for mated females without positive mating signal]. The animals were euthanized using deep CO2 followed by exsanguination.

GROSS NECROPSY
All adult animals were subjected to a full, detailed gross necropsy, which included careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents as well as organs and tissues of each animal with special emphasis on reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs from all animals (terminally sacrificed, dead and moribund sacrificed) at the scheduled sacrifices were trimmed off any adherent tissue and fat, as appropriate and weighed wet as soon as possible to avoid drying: adrenals, brain, epididymides, heart, kidneys, liver, lungs, ovaries, pituitary, prostate (all main group males), seminal vesicles with coagulation glands (all main group males), spleen, testes, thymus, thyroid and parathyroid glands and uterus with cervix.

Relative organ weights were calculated based on terminal fasting body weight.

The uterus of each dam was observed for implantation sites and recorded the count for comparison of number of implantation sites with the number of pups born for each litter to calculate the post-implantation loss. The uterus of females with no visible implantation sites (non-pregnants) was immersed in 10% v/v Ammonium Sulphide stain under fume hood to visualize the implantation sites that might have undergone very early resorptions.

Histopathological examination was conducted on the following organs and tissues collected from the vehicle control and high dose group animals: adrenals, brain, caecum, colon, cowper's glands, duodenum, epididymides, eyes with optic nerve, femur bone with marrow and joint, glans penis, heart, ileum with Peyer's patches, jejunum, kidneys, levator ani plus bulbocavernosus muscle complex, liver, lungs, lymph nodes (mandibular and mesenteric), mammary gland, ovaries, pituitary, prostate (all main group males), rectum, sciatic nerve, seminal vesicles with coagulation gland (all main group males), skeletal muscle, spinal cord, spleen, stomach, testes, thymus, thyroid and parathyroid glands, trachea, urinary bladder, uterus with cervix and vagina.

All organs and tissue samples were processed, embedded in paraffin, sectioned at a thickness of 3 to 5 micrometers and stained with hematoxylin and eosin. The testes were sectioned at 3 to 4 µm and stained with hematoxylin and eosin stain and also with Periodic Acid-Schiff (PAS) and hematoxylin stain. PAS stain aided spermatogenesis evaluation.

During histopathology special emphasis was placed on stages of spermatogenesis in male gonads and histopathology of interstitial testicular cell structure including all macroscopically abnormal tissues of all animals, regardless of whether died during the experiment or sacrificed at termination.

Statistics:

All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05).
The weekly body weights and food consumption data of non-pregnant females generated during gestation period was excluded for mean calculation and statistical analysis.

The statistical analysis was followed, but not limited to the parameters, as mentioned below.
Parametric - One-way ANOVA with Dunnett's post test:
• Body weight (weekly body weights and gestation/lactation body weights)
• Percent change in body weight (weekly body weights and gestation/lactation body weights)
• Food consumption (weekly and gestation/lactation)
• Hematology
• Clinical chemistry
• Urinalysis
• Absolute/relative organ weights
• Functional observation parameters

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Main group and recovery group:
- 450 mg/kg bw/day: all animals of both sexes did not reveal any clinical signs till day 32 of the treatment period. However, some of the animals were noted with below mentioned test item-related clinical signs during daily clinical signs observations and weekly detailed clinical examinations from day 33. These observations were continued till termination for main groups. The animals from recovery group were recovered from the signs and found normal during recovery period.
- In main group males, two animals were noted with clinical signs of systemic toxicity during treatment period. Among these, one animal was noted with perinasal staining from day 33 and the second animal from day 34. Both animals were noted with rough hair coat along with perinasal staining from day 35 and continued till termination (i.e. day 47).
- In main group females, one animal was noted with lethargy along with wet perineum from day 33; additionally, rough hair coat was observed from day 35 and continued till termination (i.e. day 60).
- In recovery group males, two animals were noted with clinical signs of systemic toxicity during treatment period. Among these, one animal was noted with lethargy from day 34, lethargy along with rough hair coat from day 35 and found normal during recovery period (i.e. from day 57). Another male was noted with perinasal staining from day 35 and found normal during recovery period (i.e. from day 56).
- In recovery group females, one animal was noted with perinasal staining from day 36 and found normal during recovery period (i.e. from day 55).

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1) Males until termination and females during pre-mating and mating period; non-pregnant females with and without positive mating signal during post-mating period
Main group and recovery group:
- 450 mg/kg bw/day: in males and females, the following statistically significant changes were noted when compared with vehicle control groups;
- decrease in mean body weight on days 22, 29, 36 and 43 at main group males (within the in-house historical control range, except for day 43)
- decrease in mean body weight on days 36, 43, 50 and 57 at recovery group males (within the in-house historical control range, except for day 43)
- decrease in mean body weight on days 22, 29, 36, 43, 50, 57 and 64 at recovery group females (below the in-house historical control range, except for day 22);
- decrease in percent change in mean body weight gain (%) during day 1 to 14,
1 to 22, 1 to 29, 1 to 36 and 1 to 43 at main group males (below the in-house historical control range, except for day 1 to 14);
- decrease in percent change in mean body weight gain (%) during day 1 to 14 at main group females (within the in-house historical control range)
- decrease in percent change in mean body weight gain (%) during day 1 to 22,
1 to 29, 1 to 36, 1 to 43, 1 to 50, 1 to 57 and 1 to 64 at recovery group males (below the in-house historical control range, except for day 1 to 57 and 1 to 64)
- decrease in percent change in mean body weight gain (%) during day 1 to 22,
1 to 29, 1 to 36, 1 to 43, 1 to 50, 1 to 57 and 1 to 64 at recovery group females (below the in-house historical control range).

Along with above mentioned statistically significant changes, toxicologically relevant decrease in mean body weight and percent change in mean body weight gain with respect to day 1 was noted at main group/recovery group males and females starting from week 2 till the end of the treatment period when compared with vehicle control groups. These decreases are within the in-house historical control range for mean weekly body weight during week 2 from recovery group females and for mean percent change in body weight gain during week 2 from recovery group males and females.

These changes are considered as test item-related due to noted test item-related clinical signs of systemic toxicity in some of the animals, reduced food consumption and effects on reproductive/developmental end points at this dose level.

2) Gestation period
Main group:
- 450 mg/kg bw/day: decrease in mean gestational body weight and percent change in mean gestational body weight gain was noted throughout the gestation period when compared with the vehicle control group. These changes are statistically significant on gestation day gestation days 7, 14 and 20 for mean gestational body weight (within the in-house historical control range) and statistically significant during gestation day 7 to 14 and 14 to 20 for mean percent change in gestational body weight gain (below the in-house historical control range for gestation day 7 to 14 and within the range for gestation day 14 to 20) when compared with the vehicle control group.

These changes are considered as test item-related due to noted treatment related reduced mean body weight/percent change in body weight gain during pre-mating period and reduced food consumption during pre-mating period at this dose level. These changes can also be correlated with continued treatment related reduced body weights, food consumption and effects on reproductive/developmental end points.

3) Lactation period
Main groups:
- 450 mg/kg bw/day: decrease in mean lactation body weight and percent change in mean lactation body weight gain was noted throughout the lactation period when compared with the vehicle control group. These changes are statistically significant on lactation days 7 and 13 for mean lactation body weight (within the in-house historical control range) and statistically significant during lactation days 4 to 7 and 7 to 13 for mean percent change in lactation body weight gain when compared with the vehicle control group (below the in-house historical control range).

These changes are considered as test item-related due to noted treatment related reduced mean body weight/percent change in body weight gain during pre-mating and gestation periods, reduced food consumption and also due to effects on reproductive/developmental end points at this dose level.

4) Terminal body weight
Main group:
- 450 mg/kg bw/day: in males and females, test item-related statistically significant decrease (p < 0.05) in mean terminal body weight was noted when compared with vehicle control group.

Recovery group:
450 mg/kg bw/day: in males and females, test item-related statistically significant decrease (p < 0.05) in mean terminal body weight was noted when compared with vehicle control group.

Please also refer to the field "Attached background material" below.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

1) Premating period (males and females) and postmating (males and non-pregnant females with and without positive mating signal)
Main group and recovery group:
- 450 mg/kg bw/day: in males and females, the following statistically significant changes in mean food consumption were noted when compared with vehicle control groups;
- decrease during week 2 (pre-mating period) and week 6 (post-mating period) at main group males (within the in-house historical control range);
- decrease during week 2 (premating period) at main group females (within the in-house historical control range);
- decrease during weeks 4, 5, 6, 7 and 8 at recovery group males (within the in-house historical control range);
- decrease during weeks 2 and 3 at recovery group females (within the in-house historical control range).

Along with above mentioned statistically significant changes, toxicologically significant decrease in mean food consumption was noted at recovery group males and females starting from week 2 till the end of the treatment period when compared with vehicle control groups. However, a slight improvement was noted during recovery period at these dose groups and all the obtained mean values were within the in-house historical control range.

These changes are considered as test item-related due to noted treatment related clinical signs of systemic toxicity in some of the animals, reduced mean body weight, reduced percent change in mean body weight gain and effects on reproductive/developmental end points at these dose levels.

2) Gestation period
Main group:
- 450 mg/kg bw/day: statistically significant decrease in mean food consumption was noted during entire gestation period measured between gestation days 0 to 7, 7 to 14 and 14 to 20 when compared with the vehicle control group (within the in-house historical control range).

These changes are considered as test item-related due to noted treatment related reduced mean body weight/percent change in body weight gain during pre-mating period and reduced food consumption during pre-mating period at this dose level. These changes can also be correlated with continued test item-related reduced body weights and food consumption and also with effects on reproductive/developmental end points at this dose level.

3) Lactation period
Main groups:
- 450 mg/kg bw/day: statistically significant decrease in mean food consumption was noted during entire lactation period measured between lactation days 1 to 4, 4 to 7 and 7 to 13 when compared with the vehicle control group (within the in-house historical control range).

These changes are considered as test item-related due to noted treatment related reduced mean body weight/percent change in body weight gain during pre-mating and gestation periods, reduced food consumption during pre-mating and gestation periods and also due to effects on reproductive/developmental end points at this dose level.

Please also refer to the field "Attached background material" below.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Main group:
- 450 mg/kg bw/day: in males, the following statistically significant changes were noted when compared with vehicle control groups:

- decrease in mean absolute weight of testes (below the in-house historical control range), absolute weight of epididymides (below the in-house historical control range), absolute and relative weight of prostate (below the in-house historical control range) and absolute and relative seminal vesicles with coagulating glands (within in-house historical control range for absolute weight and below the in-house historical control range for relative weight) in main group males

Recovery group:
- 450 mg/kg bw/day: in males, the following statistically significant changes were noted when compared with vehicle control groups:
- decrease in mean absolute weight of seminal vesicles with coagulating glands in recovery group males (within the in-house historical control range);

The noted changes in absolute/relative weights of testes, epididymis, prostate and seminal vesicles with coagulating glands can be considered test item-related due to observed gross or histopathological changes in accessary sex organs and also due to noted effects on fertility of males at this dose level.

Please also refer to the field "Attached background material" below.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Main group.
- 450 mg/kg bw/day: test item-related gross pathological findings in form of small sized prostate and seminal vesicle with coagulating gland was noted from 9/12 males. These gross pathological changes were not recorded/found in the in-house historical control group animals of same sex and strain. There were no gross pathological changes noted in the recovery dose group (450 mg/kg bw/day) at the end of the recovery period.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- 450 mg/kg bw/day: microscopically, test item-related changes were observed in prostate (12/12 males) and seminal vesicle (6/12 males) but coagulating glands were within normal limits. The histopathological change observed in prostate and seminal vesicles was atrophy of acinar cell type. It was characterised by multifocal areas of minimal to moderate reduction in acinar size of prostate with reduction or absence of luminal secretions. Similarly, the atrophy in seminal vesicle was characterised by focal area of minimal to mild reduction in vesicle lumen with reduction or absence of secretions and increased epithelial folds.

The incidence of atrophy of prostate and seminal vesicles was noted only in main group males and was considered as test item-related effect. These noted microscopic observations from main group males are not in the in-house historical control findings. This change was found to be reversible at the end of the 14-day recovery period.

Please also refer to the field "Attached background material" below.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

CLINICAL SIGNS
Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no clinical signs of toxicity noted in the daily clinical signs observations in any of the animals of both sexes throughout the experimental period. Furthermore, the detailed clinical examination conducted once in a week did not reveal any changes in any of the animals of both sexes.

Recovery groups:
- 200 mg/kg bw/day: there were no clinical signs of toxicity noted in the daily clinical signs observations in any of the animals of both sexes throughout the experimental period. Furthermore, the detailed clinical examination conducted once in a week did not reveal any changes in any of the animals of both sexes.

MORTALITY
Main groups:
- 50, 100, 200 and 450 mg/kg bw/day: there were no increased mortality or morbidity noted in any of the animals of both sexes throughout the experimental period.

Recovery groups:
- 200 and 450 mg/kg bw/day: there were no increased mortality or morbidity noted in any of the animals of both sexes throughout the experimental period.

BODY WEIGHT AND WEIGHT CHANGES
1) Males until termination and females during pre-mating and mating period; non-pregnant females with and without positive mating signal during post-mating period

Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 in both sexes throughout the experimental period.

Recovery groups:
- 200 mg/kg bw/day: there were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 in both sexes throughout the experimental period.

2) Gestation period
Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no changes noted in mean gestational body weight and percent change in mean gestational body weight gain at the dose groups when compared with the vehicle control group.

3) Lactation period
Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no changes noted in mean lactation body weight and percent change in mean lactation body weight gain at the tested dose groups when compared with the vehicle control group.

4) Terminal body weight
Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no changes noted in mean terminal body weights at the tested dose groups of both sexes when compared with vehicle control group.

Recovery group:
- 200 mg/kg bw/day: there was no change noted in mean terminal body weight at the tested dose group of both sexes when compared with vehicle control group.

FOOD CONSUMPTION
1) Premating period (males and females) and postmating (males and non-pregnant females with and without positive mating signal)

Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no changes noted in mean food consumption in both sexes during pre-mating and post-mating periods when compared with the vehicle control group.

Recovery groups:
- 200 mg/kg bw/day: there were no changes noted in mean food consumption in both sexes during pre-mating and post-mating periods when compared with the vehicle control group.

2) Gestation period
Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no changes noted in mean food consumption during gestation period at the tested dose groups when compared with the vehicle control group.

3) Lactation period
Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no changes noted in mean food consumption during lactation period at the tested dose groups when compared with the vehicle control group.

OPHTALMOLOGICAL FINDINGS
Main groups:
- 0, 50, 100, 200 and 450 mg/kg bw/day: there were no ocular changes observed in any of the animals of both sexes during ophthalmological examination (pre-treatment and shortly before scheduled sacrifice).

Recovery groups:
- 0, 200 and 450 mg/kg bw/day: there were no ocular changes observed in any of the animals of both sexes during ophthalmological examination (pre-treatment and shortly before scheduled sacrifice)

HAEMATOLOGICAL FINDINGS
Main groups:
- 50, 100, 200 and 450 mg/kg bw/day: there were no test item-related changes noted in mean haematological parameters at all tested main groups of both sexes when compared with the vehicle control group.

Recovery groups:
200 and 450 mg/kg bw/day: there were no test item-related changes noted in mean haematological parameters at all tested recovery groups of both sexes when compared with the vehicle control group.

Main groups and recovery groups:
The following statistically significant changes were noted in haematological parameters when compared with vehicle control groups.
- decrease in mean corpuscular hemoglobin concentrations in main group males of the 50, 200 and 450 mg/kg bw/day dose levels (within the in-house historical control range);
- increase of mean neutrophils (%) in main group males of the 200 mg/kg bw/day dose level (within the in-house historical control range);
- increase in the mean activated prothrombin time in main group females of the 100 mg/kg bw/day dose level (within the in-house historical control range);
- increase in the mean absolute lymphocyte count in recovery group males of the 200 mg/kg bw/day dose level (within the in-house historical control range);
- increase in the mean reticulocyte count (%) and mean prothrombin time in recovery group females of the 450 mg/kg bw/day dose level (within the in-house historical control range).

These changes are considered as incidental and unrelated to treatment as the changes did not occurr in a dose dependent manner and also all the obtained mean values for these significant changes are within in-house historical control range of same species and strain.

CLINICAL BIOCHEMISTRY FINDINGS
Main groups:
- 50, 100, 200 and 450 mg/kg bw/day: there were no test item-related changes noted in mean clinical chemistry parameters at all tested main groups of both sexes when compared with the vehicle control groups.

Recovery groups:
- 200 and 450 mg/kg bw/day: there were no test item-related changes noted in mean clinical chemistry parameters at all tested recovery groups of both sexes when compared with the vehicle control groups.

Main groups and recovery groups:
The following statistically significant changes were noted in clinical chemistry parameters when compared with vehicle control groups.
- increase in mean creatinine levels in main group males of the 50 and 450 mg/kg bw/day dose levels (within the in-house historical control range);
- increase in mean calcium levels in main group of the 50 and 450 mg/kg bw/day dose levels (within the in-house historical control range);
- decrease in mean cholinesterase levels in main group of the 200 mg/kg bw/day dose level (within the in-house historical control range);
- increase in mean alanine aminotransferase in main group females of the 100 mg/kg bw/day dose level (within the in-house historical control range);
- decrease in mean alkaline phosphatase levels in recovery group females of the 450 mg/kg bw/day dose level (within the in-house historical control range).

These changes are considered as incidental and unrelated to treatment as the changes did not occurre in a dose dependent manner and also all the obtained mean values for these significant changes are within in-house historical control range of same species and strain.

URINALYSIS
Main groups:
- 50, 100, 200 and 450 mg/kg bw/day: there were no test item-related changes noted in mean urinalysis parameters at all tested main group males when compared with the vehicle control group.

- 450 mg/kg bw/day: statistically significant decrease in mean urine volume was observed in males (4.5 ± 1.5 mL; p <0.05) when compared with vehicle control group (9.4 ± 2.6 mL). This noted reduction in mean value is below the in-house historical control range (7.17 ± 1.98 mL; acceptable limit: 5.19 - 9.14 mL). However, the noted reduction was considered as incidental and unrelated to treatment as the rest of the urine physical, chemical and microscopic parameters were unaffected at this dose level.

Recovery groups:
- 200 and 450 mg/kg bw/day: there were no test item-related changes noted in mean urinalysis parameters in all recovery groups of both sexes when compared with the vehicle control group.

BEHAVIOUR (FUNCTIONAL FINDINGS)
Main groups and recovery groups:
- 50, 100, 200 and 450 mg/kg bw/day: the neurological/functional observations did not reveal any changes in any of the animals of both sexes from all the tested dose groups performed towards end of the dosing period for main groups (on day 45 for males and on lactation day 13 for females/dams) and performed towards end of recovery period for recovery groups (day 65).

There were no test item-related changes noted in mean fore/hind limb grip strengths, mean motor activity assessments and mean hind limb foot splay at all tested dose groups of both sexes when compared with vehicle control groups during conduct of neurological/functional examinations.

Main group:
- 100 mg/kg bw/day: the noted statistically significant increase in mean movement counts during motor activity assessment in males when compared with vehicle control group is considered as incidental and toxicologically not relevant and also the obtained mean value is within in-house historical control range.

Recovery groups:
200 and 450 mg/kg bw/day: the noted statistically significant decrease in mean number of grooming in females of the 200 mg/kg bw/day group and statistically significant decrease in mean hind limb grip strength in females of the 450 mg/kg bw/day group when compared with vehicle control group can be considered as incidental as there were no such changes noted in main groups and also no effects noted in forelimb grip strength of same group animals conducted at similar time. Also, the noted mean value for mean hind limb grip strength in the females of the 450 mg/kg bw/day group was within in-house historical control range.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN WEIGHT / BODY WEIGHT RATIOS
Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no test item-related changes noted in mean absolute and relative organ weights at the tested dose groups of both sexes when compared with vehicle control group.

- 50 and 100 mg/kg bw/day: the noted statistically significant increase (p < 0.05) in mean absolute weight of spleen (within the in-house historical control data normal range, but above the acceptable limits) and statistically significant decrease (p < 0.05) in mean absolute and relative weight of ovaries in females (Absolute weight: within the in-house historical control range for 100 mg/kg bw/day group; below the in-house historical control range for 50 mg/kg bw/day group; relative weight: below the in-house historical control range for both groups) when compared with vehicle control group are considered as incidental and unrelated to treatment as there were no gross pathological changes noted in any of these organs during necropsy from any of the animals of these dose groups and there is no dose-response relationship.

- 450 mg/kg bw/day: in males and females, the following statistically significant changes were noted when compared with vehicle control group:
- increase in mean relative weight of adrenals, brain, liver and lungs in main group males (within the in-house historical control range);
- decrease in mean absolute weight of ovaries and thyroid along with parathyroid in main group females (within the in-house historical control range)
- increase in mean relative weight of thymus, spleen, uterus with cervix, heart, brain, liver, lungs, pituitary gland and thyroid along with parathyroid in main group females (within the in-house historical control range, except for spleen and lungs);

Recovery group:
- 200 mg/kg bw/day: there were no test item-related changes noted in mean absolute and relative organ weights of both sexes when compared with vehicle control group.

- 450 mg/kg bw/day: in males and females, the following statistically significant changes were noted when compared with vehicle control group:
- increase in mean relative weight of thymus in recovery group males (within the in-house historical control range);
- increase in mean relative weight of kidneys, brain and liver in recovery group females (within the in-house historical control range)

The observed changes in mean absolute/relative weights of organs such as, spleen, ovaries, kidneys, uterus with cervix, heart, thymus, adrenals, brain, liver, lungs, pituitary gland and thyroid along with parathyroid are considered as incidental in both sexes as there were no gross or histopathological changes noted at this dose level during necropsy or microscopic examinations and also the obtained values are within in-house historical control ranges.

Please also refer to the field "Attached background material" below.

HISTOPATHOLOGICAL FINDINGS - NON-NEOPLASTIC
- 0 and 450 mg/kg bw/day: a few other microscopic findings were observed in the study such as ultimobranchial cyst in thyroid, cystic tubule and infiltration of mononuclear cells in interstitium in kidneys and accessory cortical tissue in adrenals. However, these findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats of this age (Elizabeth McInnes, 2012)*.

GROSS PATHOLOGICAL FINDINGS
Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no gross pathological changes noted at the dose groups during conduct of necropsy.

Recovery group:
- 200 mg/kg bw/day: there were no gross pathological changes noted at the dose group during conduct of necropsy.

ENDOCRINE FINDINGS
Main groups and recovery groups:
- 50, 100, 200 and 450 mg/kg bw/day: there were no test item-related changes noted in serum T3, T4 and TSH levels at any of the tested main and recovery groups from both sexes when compared with vehicle control groups.

- 50, 200 and 450 mg/kg bw/day: the statistically significant increase in mean serum T3 levels in females of the 50 mg/kg bw/day dose level (main group) and statistically significant increase in mean serum TSH levels in females of the 200 mg/kg bw/day dose level (main group) are considered as incidental and unrelated to treatment as the changes are not dose dependent and also the obtained values are within in-house historical control range of same species and strain.

Please also refer to the field "Attached background material" below.

*Reference:
- Elizabeth F. McInnes, 2012, Background lesions in Laboratory animals: A color Atlas, Saunders, Elsevier, Edinburgh.

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

not specified

Conclusions:

In a current combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, barium 4-dodecylphenolate was administered via gavage to groups of male and female Sprague-Dawley rats (n = 12 animals/sex/group) at dose levels of 50, 100, 200 and 450 mg/kg bw/day. The test item was administered once daily for seven days during premating period, mating period, pregnancy and lactation. A vehicle (corn oil) control group was run concurrently. In addition, three recovery groups with 5 males and 5 females each (0, 200 and 450 mg/kg bw/day) were also run concurrently to determine the reversibility of the test item related effects.

During the observations of the parental male and female rats, no test item-related effects were observed for mortality, opthalmology, haematology, clinical chemistry including serum thyroid hormone levels (T3, T4 and TSH), urinalysis, or neurobehaviour.

At the 450 mg/kg bw/day dose level, some male and female animals were noted with test item-related clinical signs such as perinasal staining, rough coat, lethargy, wet perineum. Animals of the recovery group fully recovered from these clinical signs and were found normal during the recovery period. Furthermore, animals of the 450 mg/kg bw/day dose group were also noted with test-item related reduction in body weight and body weight gain as well as in food consumption. Test item-related reduced absoute/relative weights of testes, epididymis, prostate and seminal vesicles with coagulating glands were also noted at this high dose level. Test item-related gross pathological changes in form of small sized prostate and seminal vesicle with coagulating gland and microscopically, test item-related acinar atrophy in the prostate and seminal vesicles were noted in males at the 450 mg/kg bw/day dose level. The recovery group animals of the 450 mg/kg bw/day dose group recovered during the recovery period and were found normal at termination.

Based on the substance-related effects in the high dose group of 450 mg/kg bw/day, the no observed adverse effect level (NOAEL) of this study is 200 mg/kg bw/day for males and females.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed 2019-09-16

Limit test:

no

Justification for study design:

not applicable

Test material

Test material information

Constituent 1

Reference substance name:

Barium 4-dodecylphenolate

EC Number:

300-141-0

EC Name:

Barium 4-dodecylphenolate

Cas Number:

93922-04-4

Molecular formula:

C18H30O.1/2Ba

IUPAC Name:

barium 4-dodecylphenolate

Test material form:

solid

Details on test material:

- State of aggregation: brownish, inhomogeneous solid with a chewy texture

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (21 to 29°C), in a dark and dry room in the original container

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:SD

Details on species / strain selection:

Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Bioneeds India Private Limited (in-house bred animals)
- Females nulliparous and non-pregnant: yes

- Age at study initiation (treatment day 1):
Males: 87 to 95 days (12 to 13 weeks)
Females: 87 to 95 days (12 to 13 weeks)

- Weight at study initiation (treatment day 1):
Males: 271.93 to 326.31 g (main and recovery groups)
Females: 222.79 to 247.13 g (main and recovery groups)

- Housing: housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube; bedding material: clean sterilized paddy husk
- Acclimatization: maximum of three animals/sex were housed together
Main group animals:
- Pre-mating: 2 animals/sex/group were housed together
- Cohabitation Period (mating) - 1 male / 1 female of the same group were housed together
- Post-mating: males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as nesting material for main group females from gestation day 20 onwards. Lactating females were housed individually with their offspring.
Recovery Group Animals: maximum of three animals/cage per group of the same sex were housed.

- Diet (ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (ad libitum): deep bore-well water passed through reverse osmosis unit

- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19.8 to 23.2 °C
- Relative humidity: 49 to 64 %
- Air changes: 12 to 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulations were freshly prepared before dose administration on each treatment day and they were administered as soon as possible after preparation.

The required quantity of test item was weighed into a clean glass beaker. A little volume of vehicle was added into the beaker and mixed well with glass rod and then placed on a hot plate magnetic stirrer with a set temperature of 40 to 60°C. The formulation was allowed to stir under this condition by adding little quantity of vehicle to facilitate homogenous sampling. After confirmation of uniform suspension of formulation, the entire content was transferred into the measuring cylinder and the beaker was rinsed using a small volume of vehicle and the volume was transferred to measuring cylinder. The rinsing procedure was repeated until entire quantity of the test item formulation was transferred into a measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get a desired concentration of test item. After that, the entire quantity was transferred to beaker, allow for stirring by placing on a hot plate magnetic stirrer with a set temperature of 40 to 60°C until uniform suspension of test item formulation was obtained.

The test item formulations were allowed to cool down at room temperature prior to administration. The test item formulations were maintained under stirring conditions using magnetic stirrer to maintain homogeneity of the test item formulations during test item administration.

Administration volume: 4 mL/kg bw
The actual dose volume for each animal was calculated based on the most recent body weight.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
The test item was not soluble in distilled water and could not be suspended in
0.5 % w/v carboxy methyl cellulose at the concentration of 25 mg/mL (the highest dose concentration considering an equivolume of 10 mL/kg body weight). The test item formed a uniform suspension with corn oil at the concentration of 125 mg/mL (the highest dose concentration considering equivolume of 4 mL/kg body weight) as per in-house miscibility test results.
Corn oil was used as vehicle for test item formulations in the range finding study. For consistency, this is the preferred vehicle for the present study.
Corn oil is universally accepted and routinely used vehicle in oral toxicity studies. Hence, corn oil was selected as vehicle for test item formulations.

- Manufacturer: MP Biomedicals
- Lot/batch no.: L32011001
- Expiry date: February 2023

Details on mating procedure:

NOTE: the recovery group animals were not used for mating and, consequently, not used for the assessment of reproduction/developmental toxicity.

- M/F ratio per cage: 1 male / 1 female

- Length of cohabitation: female was placed with the same male until presence of sperm in the vaginal smear and the occurence of a vaginal plug or up to 14 days.

- Proof of pregnancy: every morning, the vaginal smear of each female was examined for presence of sperm. Day ‘0’ pregnancy was confirmed by the presence of sperm in the vaginal smear and a vaginal plug.

- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.

- Further matings after two unsuccessful attempts: no
The females which were not positive for presence of sperm in the vaginal smear and a vaginal plug after second opportunity with a proven male were treated for further 24 days and sacrificed on 25th day after the last day of the mating period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of test item in dose formulations were established. The test item barium 4-dodecylphenolate was stable for 48 hours at room temperature in corn oil at the concentrations of 5 mg/mL and 150 mg/mL.

In addition, homogeneity and dose formulation analysis for dose concentration verification was conducted. For the determination of the test item concentration in the prepared test item formulations, samples were taken and stored at -20°C ± 10% until analysis. Sampling and analysis of formulations was performed during week 1, week 4 and week 7 of the treatment. The samples were collected in duplicates from top, middle and bottom layers from control (vehicle), low (50 mg/kg bw/day), low intermediate (100 mg/kg bw/day), high intermediate (200 mg/kg bw/day) and high dose concentrations (450 mg/kg bw/day) and in duplicates from single layer from vehicle control.

One set of aliquots of each formulation was analyzed. The second aliquot was stored as a backup purpose at established stability conditions. Formulations were considered acceptable, since the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is less than 10%.

The following ranges were determined for the treatment groups:
Recovery of formulations (%).
50 mg/kg bw/day: 93.18 % - 100.15 % (% RSD: 0.41 - 2.44)
100 mg/kg bw/day: 92.93 % - 96.09 % (% RSD: 0.60 - 3.18)
200 mg/kg bw/day: 93.80 % - 107.04 % (% RSD: 0.30 - 1.62)
450 mg/kg bw/day: 94.46 % - 106. 47 % (% RSD: 0.53 - 0.84)

Duration of treatment / exposure:

1) Main group:
- males: two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 46 days of treatment)
- females (pregnant/littered): two-week pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 (ranging from a total period of 49 to 67 days)
-females (mated but non-pregnant): two-week pre-mating period, during mating until confirmed as mated and further 24 days from the day of confirmation of mating (ranging from a total period of 43 to 57 days)
- females: (without positive mating signal): two-week pre-mating period, during the entire mating period i.e. 21 days and further 24 days from the day of termination of mating (with a total period of 59 days)

2) Recovery group:
- males and females: 52 days

Frequency of treatment:

once daily, 7 days/week

Details on study schedule:

not applicable

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

Main group

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Main group

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

Main group and recovery group

Doses / concentrations 4

Dose / conc.:

450 mg/kg bw/day (actual dose received)

Remarks:

Main group and recovery group

No. of animals per sex per dose:

Main groups: 12 males / 12 females
Recovery groups: 5 males / 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: for dose selection, information from an acute oral toxicity study (please refer to IUCLID section 7.2.1 Acute toxicity: oral: BaDodec_k_Niklas_2018) with barium 4-dodecylphenolate was used, since substance-specific data are limited and this test is the most valuable with regard to systemic toxicity. In this study, the No Observed Adverse Effect Level [NOAEL] of test item barium 4-dodecylphenolate is 300 mg/kg body weight. Hence, the doses of 0, 20, 100, 250 and 500 mg/kg body weight/day were selected as control, low, low mid, high mid and high dose groups for a dose range finding study performed at the laboratory. Groups of 5 male and 5 female Sprague Dawley rats were administered via gavage with the test item in corn oil at the four dose levels. A vehicle control group was run concurrently.

Results:
There were no treatment related significant changes in any of the systemic parameters in either sex noted at 20, 100 and 250 mg/kg body weight/day. However, significant reduction in body weight, food consumption and reproductive organ weights (prostate along with seminal vesicles with coagulating gland) were noted at 500 mg/kg body weight/day in males only in the dose range finding study.

Based on the results obtained from dose range finding study, the doses of 0, 100, 200, 450 and 50 mg/kg body weight were selected for the present study.

Positive control:

not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked: clinical signs and mortality/morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: day 1 before treatment and weekly thereafter during treatment

BODY WEIGHT: Yes
- Time schedule for examinations:
Main group males: at receipt, on the day of randomization, on the first day of dosing, weekly thereafter and at termination

Main group females: at receipt, on the day of randomization, on the first day of dosing, weekly thereafter during pre-mating and until confirmation of mating.

Main group females (pregnant): gestation days 0, 7, 14 and 20 and on lactation days 1, 4, 7 and 13

Main group females (non-pregnant with and without positive mating signal): once in a week.

All the females were weighed terminally on the day of sacrifice.

Recovery group animals (males and females): at receipt, on the day of randomization, on the first day of dosing, weekly thereafter and at termination.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Main group males: cage wise food consumption was measured once a week during pre-mating period (week 1 and 2) and post-mating period (week 6).

Main group females: cage wise food consumption was measured once in a week during pre-mating period coinciding with body weight recording.

Main group females (pregnant): cage wise food consumption was measured during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4, 4 to 7 and 7 to 13.

Main group females (non-pregnant with and without positive mating signal): cage wise food consumption was measured once in a week coinciding with body weight recording during the post mating period.

Food consumption was not measured for both sexes during mating period, for the pregnant females after gestation day 20 until parturition

Recovery group animals: once per week throughout the experimental period.

Average food intake per rat (g/rat/day) was calculated using the amount of food offered and left over in each cage and the number of rats per cage. Food spillage was taken into consideration.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once before treatment for all animals, at the end of the dosing period for males (shortly prior to scheduled sacrifice, i.e. day 44), during the lactation period for pregnant females (shortly prior to scheduled sacrifice, i.e. on lactation day 13) and one day prior to sacrifice for non-pregnant females (i.e. experimental day 44 to 58) of all main group animals and during the last week (i.e. day 66) for the recovery group animals
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled sacrifice (for main group males after completion of 46 days of treatment, for main group females on lactation day 14 and for recovery group animals on scheduled sacrifice (study day 67)
- Animals fasted: Yes, overnight (water was provided ad libitum)
- Anaesthetic used for blood collection: Yes, isoflurane anesthesia
- How many animals: 5 animals/sex (main groups and recovery groups)
- Parameters checked: haemoglobin concentration, haematocrit, erythrocyte count, total leukocyte count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, mean platelet volume, differential leucocytes count, reticulocyte count, absolute reticulocyte count, absolute differential leucocytes count, prothrombin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled sacrifice (for main group males after completion of 46 days of treatment, for main group females on lactation day 14 and for recovery group animals on scheduled sacrifice (study day 67)
- Animals fasted: Yes, overnight (water was provided ad libitum)
- Anaesthetic used for blood collection: Yes, isoflurane anesthesia
- How many animals: 5 animals/sex (main groups and recovery groups)
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, cholinesterase, total protein, albumin, total bilirubin, glucose, total cholesterol, creatinine, urea, blood urea nitrogen, triglycerides, phosphate, calcium, globulin (difference between total protein and albumin value-instrument calculated), albumin/ globulin ratio, bile acids, sodium, potassium, chloride,

ENDOCRINE EXAMINATION: Yes
- Time schedule for collection of blood: blood samples were collected from all main group animals (parental and pups) and recovery animals for measurement of serum thyroid hormone levels at a comparable time of the day on the following schedule:

Two pups per litter on postnatal day 4 based on the following conditions:
- preferably two female pups in order to retain more male pups for nipple retention on postnatal day 13 except in the event that removing these pups leaves no remaining females for assessment at termination.
- no pups were eliminated when the litter size dropped to below 10 pups / litter.
- only one pup was eliminated and used for blood collection for serum thyroid hormones assessments in case of only one pup available above the normal litter size

All the main group females at termination (lactation day 14). The blood was not collected from the main group females which were not littered within 24 to 26 days from the day of mating and sacrificed.

Two pups per litter (same sex) at termination (postnatal day 13)

All main group males, at termination (after completion of 46 days of treatment).

All recovery group males and females at termination.

Serum of pups was pooled per litter for T3, T4 and TSH analysis on both postnatal days 4 and 13, respectively.

- Parameters checked: triiodothyronine (T3), thyroxine (T4) and thyroid stimulating hormone (TSH)

URINALYSIS: Yes (males only)
- Time schedule for collection of urine: at termination
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water was provided ad libitum)
- How many animals: 5 males (main groups, satellite group and recovery groups) and 5 females (recovery group only)
- Parameters checked: overnight urine volume, volume, appearance, colour, blood, bilirubin, urobilinogen, ketones, protein, glucose, microalbumin, leucocytes, pH, nitrite, specific gravity and urine sediments

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Main groups: end of the dosing period for males (shortly prior to scheduled sacrifice, i.e. on day 45) and during the last week of lactation period for females (shortly prior to scheduled sacrifice, i.e. on lactation day 13)
Recovery groups: towards the end of the recovery period (shortly prior to scheduled sacrifice, i.e. on day 65)

- Dose groups that were examined: all dose groups (five animals/sex/dose group)

- Battery of functions tested:
- sensory activity: startle response, touch response, pupil response, response to nociceptive stimuli and righting reflexes were recorded.
- grip strength: hind limb and fore limb grip strength were recorded using a grip strength meter
- motor activity: motor activity was recorded using a Photoactometer/Locomotor activity monitoring system
- home cage observation: convulsions, tremors and palpebral closure and the scores were recorded
- handling observations: during removal from the cage, the animals were observed for its ease to be removed out of the cage. During handling observation, the animals were observed for the resistance, lacrimation, red and crusty deposits around eyes, nose and mouth.
- open field observations: mobility, gait, arousal, rearing, urination, defecation, stereotypes and excessive grooming in an open field arena were recorded
- neuromuscular observations: animals were dropped onto a recording paper sheet from a height of approx. 30 cm with painted paws to record the hind limb foot splay.
- physiological observation: rectal temperature was recorded using a calibrated digital thermometer

IMMUNOLOGY: No

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored during the acclimatization to evaluate the normal oestrus cyclicity (4 to 5 days). Only females with normal oestrus cyclicity were selected for the treatment. Vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa. Oestrus cyclicity was also monitored on the day of sacrifice for females.

Recovery group females were not evaluated for oestrus cyclicity.

Sperm parameters (parental animals):

Parameters examined in all parental males: testis weight and epididymis weight,

A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen or any cell or stage specificity of testicular findings.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in offspring:
- number of pups born (dead and live) and sex of pups were recorded at birth.
- individual body weight of live pups on lactation day 1 (within 24 hours of parturition), 4, 7 and 13 were recorded.
- any abnormal behaviour of pups during observations was recorded.
- anogenital distance of each pup was measured on lactation day 4 and was normalized to a measure of pup size (e.g. cube root of body weight).
- number of nipples/areolae in male pups was counted on lactation day 13

GROSS EXAMINATION OF DEAD PUPS:
Yes, dead pups were examined for gross abnormalities with particular attention to the external reproductive genitals and the findings were recorded.

Postmortem examinations (parental animals):

SACRIFICE
- males from main groups were sacrificed on experimental day 47;
- pregnant/littered females from main groups were sacrificed on lactation day 14;
- mated females without positive mating signal were sacrificed after 24 days from the day of termination of mating (four females from 450 mg/kg bw/day (main group) on experimental day 60).
- mated but non-pregnant females were sacrificed after 24 days from the last day of confirmation of mating (vehicle control (main group) - one female on experimental day 44; 100 mg/kg bw/day (main group) - one female on experimental day 49; 200 mg/kg bw/day (main group) - two females on experimental days 45 and 47 each; 450 mg/kg bw/day (main group) - three females on experimental days 47, 52 and 58 each; 50 mg/kg bw/day - one female on experimental day 44).
- all males and females from recovery groups were sacrificed on experimental
day 67.

All animals were fasted overnight, water was provided ad libitum during fasting. The next day, the body weight of all fasted animals were recorded prior to exsanguination. The vaginal smear was analyzed and the stage of oestrus cycle was recorded for all main group females on the day of necropsy [i.e. lactation day 14 for littered females, 24 days after the last day of mating for mated but non-pregnant females and on experimental day 60 for mated females without positive mating signal]. The animals were euthanized using deep CO2 followed by exsanguination.

GROSS NECROPSY
All adult animals were subjected to a full, detailed gross necropsy, which included careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents as well as organs and tissues of each animal with special emphasis on reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs from all animals (terminally sacrificed, dead and moribund sacrificed) at the scheduled sacrifices were trimmed off any adherent tissue and fat, as appropriate and weighed wet as soon as possible to avoid drying: adrenals, brain, epididymides, heart, kidneys, liver, lungs, ovaries, pituitary, prostate (all main group males), seminal vesicles with coagulation glands (all main group males), spleen, testes, thymus, thyroid and parathyroid glands and uterus with cervix.

Relative organ weights were calculated based on terminal fasting body weight.

The uterus of each dam was observed for implantation sites and recorded the count for comparison of number of implantation sites with the number of pups born for each litter to calculate the post-implantation loss. The uterus of females with no visible implantation sites (non-pregnants) was immersed in 10% v/v Ammonium Sulphide stain under fume hood to visualize the implantation sites that might have undergone very early resorptions.

Histopathological examination was conducted on the following organs and tissues collected from the vehicle control and high dose group animals: adrenals, brain, caecum, colon, cowper's glands, duodenum, epididymides, eyes with optic nerve, femur bone with marrow and joint, glans penis, heart, ileum with Peyer's patches, jejunum, kidneys, levator ani plus bulbocavernosus muscle complex, liver, lungs, lymph nodes (mandibular and mesenteric), mammary gland, ovaries, pituitary, prostate (all main group males), rectum, sciatic nerve, seminal vesicles with coagulation gland (all main group males), skeletal muscle, spinal cord, spleen, stomach, testes, thymus, thyroid and parathyroid glands, trachea, urinary bladder, uterus with cervix and vagina.

All organs and tissue samples were processed, embedded in paraffin, sectioned at a thickness of 3 to 5 micrometers and stained with hematoxylin and eosin. The testes were sectioned at 3 to 4 µm and stained with hematoxylin and eosin stain and also with Periodic Acid-Schiff (PAS) and hematoxylin stain. PAS stain aided spermatogenesis evaluation.

During histopathology special emphasis was placed on stages of spermatogenesis in male gonads and histopathology of interstitial testicular cell structure including all macroscopically abnormal tissues of all animals, regardless of whether died during the experiment or sacrificed at termination.

Postmortem examinations (offspring):

SACRIFICE
Pups which were sacrificed on postnatal day 4/13 were examined for gross abnormalities with particular attention to the external reproductive genitals and the findings were recorded.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
The thyroid along with the parathyroid was collected from one male and one female pup per litter on lactation day 13. In case of unavailability of both the sexes, the thyroid along with the parathyroid was collected from the same sex (either male or female) in a litter. The thyroid along with the parathyroid from the adults and pups were preserved in 10% v/v Neutral Buffered Formalin. The thyroid along with the parathyroid were weighed post fixation.

Statistics:

All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05).
Statistical analysis of pup body weight, anogenital distance and nipple retention were based on individual pup data, taking litter effects into account.

Data of non-pregnant females (with or without positive mating signal) were excluded for mean calculations and statistical analysis for determination of reproductive toxicity end points, but considered for systemic toxicity end points.

The weekly body weights and food consumption data of non-pregnant females generated during gestation period was excluded for mean calculation and statistical analysis.

The statistical analysis was followed, but not limited to the parameters, as mentioned below.
1) Parametric - One-way ANOVA with Dunnett's post test:
• Body weight (weekly body weights and gestation/lactation body weights)
• Percent change in body weight (weekly body weights and gestation/lactation body weights)
• Food consumption (weekly and gestation/lactation)
• Mean copulatory interval per dam
• Mean length of oestrus cylicity
• Mean gestation length per dam
• Hematology
• Clinical chemistry
• Urinalysis
• Absolute/relative organ weights
• Functional observation parameters
• Serum T4, T3 and TSH values (adults and pups)
• Mean pup weight (male/female) per dam
• Mean anogenital distance ratio of pups (male/female) per dam

2) Non Parametric – Kruskal-Wallis
• No. of implantations/dam
• No. of live or dead or cannibalized pups/dam at birth and during lactation period
• Sex ratio (m/f)/dam at birth and during lactation period
• Litter size at birth and during lactation period
• Number and percent of pre-natal loss/dam
• Number and percent of post-natal loss/dam

3) Cross Tabs - Chi-square test
• Pregnancy rate
• No. of dams with/without live pups per group
• No. of dams with/without dead pups per group
• No. of dams with/without resorptions per group

Reproductive indices:

- Male mating index (%) = (No. of males with evidence of mating /Total no. of males used for mating) x 100
- Male fertility index (%) = (No. of males siring a litter / Total no. of males mated) x 100
- Female mating index (%) = (No. of females with evidence of mating (or confirmed pregnancy) / Total no. of females used for mating) x 100
- Female fertility index (%) = (No. of females with confirmed pregnancy / Total no. of females used for mating) x 100
- Precoital interval (Days) = Date of confirmation mating – Date of initiation of cohabitation
- Mean gestational length = Date of parturition – Date of positive evidence of mating (GD 0)
- Gestation index (%) = (number of females with live born / number of females with evidence of pregnancy) x 100
- Mean number of implantations (%) = (Sum of number of implantations in the group / Total no. of dams in the group) x 100
- Pre-natal Loss/post-implantation loss (%) = (No. of Implantations - No. of Viable pups / No. of Implantations) x 100
- Mean Litter Size per Group = Total number of pups delivered (live and stillborn) /
Number of dams that delivered

Offspring viability indices:

- Post-natal loss on lactation day 4/13 (No.) = Live births - pups alive at post-natal day 4/13
- Live birth index (%) per dam = (Number of pups born alive / Total number of pups born) x 100
- Pup survival index (%) on lactation day 4 = (Total number of live pups (on lactation day 4) / Number of pups born) x 100
- Sex ratio (m/f) = Number of male offspring / Number of female offspring
- Percentage of male/female offspring (%) = (Number of male/female offspring /
Total number of offspring) x 100
- Ano-genital Distance to cube root of the body weight Ratio (AGD ratio) = (Ano-genital Distance (mm) / Cube root of the body weight) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Main group and recovery group:
- 450 mg/kg bw/day: all animals of both sexes did not reveal any clinical signs till day 32 of the treatment period. However, some of the animals were noted with below mentioned test item-related clinical signs during daily clinical signs observations and weekly detailed clinical examinations from day 33. These observations were continued till termination for main groups. The animals from recovery group were recovered from the signs and found normal during recovery period.
- In main group males, two animals were noted with clinical signs of systemic toxicity during treatment period. Among these, one animal was noted with perinasal staining from day 33 and the second animal from day 34. Both animals were noted with rough hair coat along with perinasal staining from day 35 and continued till termination (i.e. day 47).
- In main group females, one animal was noted with lethargy along with wet perineum from day 33; additionally, rough hair coat was observed from day 35 and continued till termination (i.e. day 60).
- In recovery group males, two animals were noted with clinical signs of systemic toxicity during treatment period. Among these, one animal was noted with lethargy from day 34, lethargy along with rough hair coat from day 35 and found normal during recovery period (i.e. from day 57). Another male was noted with perinasal staining from day 35 and found normal during recovery period (i.e. from day 56).
- In recovery group females, one animal was noted with perinasal staining from day 36 and found normal during recovery period (i.e. from day 55).

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1) Males until termination and females during pre-mating and mating period; non-pregnant females with and without positive mating signal during post-mating period
Main group and recovery group:
- 450 mg/kg bw/day: in males and females, the following statistically significant changes were noted when compared with vehicle control groups;
- decrease in mean body weight on days 22, 29, 36 and 43 at main group males (within the in-house historical control range, except for day 43)
- decrease in mean body weight on days 36, 43, 50 and 57 at recovery group males (within the in-house historical control range, except for day 43)
- decrease in mean body weight on days 22, 29, 36, 43, 50, 57 and 64 at recovery group females (below the in-house historical control range, except for day 22);
- decrease in percent change in mean body weight gain (%) during day 1 to 14,
1 to 22, 1 to 29, 1 to 36 and 1 to 43 at main group males (below the in-house historical control range, except for day 1 to 14);
- decrease in percent change in mean body weight gain (%) during day 1 to 14 at main group females (within the in-house historical control range)
- decrease in percent change in mean body weight gain (%) during day 1 to 22,
1 to 29, 1 to 36, 1 to 43, 1 to 50, 1 to 57 and 1 to 64 at recovery group males (below the in-house historical control range, except for day 1 to 57 and 1 to 64)
- decrease in percent change in mean body weight gain (%) during day 1 to 22,
1 to 29, 1 to 36, 1 to 43, 1 to 50, 1 to 57 and 1 to 64 at recovery group females (below the in-house historical control range).

Along with above mentioned statistically significant changes, toxicologically relevant decrease in mean body weight and percent change in mean body weight gain with respect to day 1 was noted at main group/recovery group males and females starting from week 2 till the end of the treatment period when compared with vehicle control groups. These decreases are within the in-house historical control range for mean weekly body weight during week 2 from recovery group females and for mean percent change in body weight gain during week 2 from recovery group males and females.

These changes are considered as test item-related due to noted test item-related clinical signs of systemic toxicity in some of the animals, reduced food consumption and effects on reproductive/developmental end points at this dose level.

2) Gestation period
Main group:
- 450 mg/kg bw/day: decrease in mean gestational body weight and percent change in mean gestational body weight gain was noted throughout the gestation period when compared with the vehicle control group. These changes are statistically significant on gestation day gestation days 7, 14 and 20 for mean gestational body weight (within the in-house historical control range) and statistically significant during gestation day 7 to 14 and 14 to 20 for mean percent change in gestational body weight gain (below the in-house historical control range for gestation day 7 to 14 and within the range for gestation day 14 to 20) when compared with the vehicle control group.

These changes are considered as test item-related due to noted treatment related reduced mean body weight/percent change in body weight gain during pre-mating period and reduced food consumption during pre-mating period at this dose level. These changes can also be correlated with continued treatment related reduced body weights, food consumption and effects on reproductive/developmental end points.

3) Lactation period
Main groups:
- 450 mg/kg bw/day: decrease in mean lactation body weight and percent change in mean lactation body weight gain was noted throughout the lactation period when compared with the vehicle control group. These changes are statistically significant on lactation days 7 and 13 for mean lactation body weight (within the in-house historical control range) and statistically significant during lactation days 4 to 7 and 7 to 13 for mean percent change in lactation body weight gain when compared with the vehicle control group (below the in-house historical control range).

These changes are considered as test item-related due to noted treatment related reduced mean body weight/percent change in body weight gain during pre-mating and gestation periods, reduced food consumption and also due to effects on reproductive/developmental end points at this dose level.

4) Terminal body weight
Main group:
- 450 mg/kg bw/day: in males and females, test item-related statistically significant decrease (p < 0.05) in mean terminal body weight was noted when compared with vehicle control group.

Recovery group:
450 mg/kg bw/day: in males and females, test item-related statistically significant decrease (p < 0.05) in mean terminal body weight was noted when compared with vehicle control group.

Please also refer to the field "Attached background material" below.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

1) Premating period (males and females) and postmating (males and non-pregnant females with and without positive mating signal)
Main group and recovery group:
- 450 mg/kg bw/day: in males and females, the following statistically significant changes in mean food consumption were noted when compared with vehicle control groups;
- decrease during week 2 (pre-mating period) and week 6 (post-mating period) at main group males (within the in-house historical control range);
- decrease during week 2 (premating period) at main group females (within the in-house historical control range);
- decrease during weeks 4, 5, 6, 7 and 8 at recovery group males (within the in-house historical control range);
- decrease during weeks 2 and 3 at recovery group females (within the in-house historical control range).

Along with above mentioned statistically significant changes, toxicologically significant decrease in mean food consumption was noted at recovery group males and females starting from week 2 till the end of the treatment period when compared with vehicle control groups. However, a slight improvement was noted during recovery period at these dose groups and all the obtained mean values were within the in-house historical control range.

These changes are considered as test item-related due to noted treatment related clinical signs of systemic toxicity in some of the animals, reduced mean body weight, reduced percent change in mean body weight gain and effects on reproductive/developmental end points at these dose levels.

2) Gestation period
Main group:
- 450 mg/kg bw/day: statistically significant decrease in mean food consumption was noted during entire gestation period measured between gestation days 0 to 7, 7 to 14 and 14 to 20 when compared with the vehicle control group (within the in-house historical control range).

These changes are considered as test item-related due to noted treatment related reduced mean body weight/percent change in body weight gain during pre-mating period and reduced food consumption during pre-mating period at this dose level. These changes can also be correlated with continued test item-related reduced body weights and food consumption and also with effects on reproductive/developmental end points at this dose level.

3) Lactation period
Main groups:
- 450 mg/kg bw/day: statistically significant decrease in mean food consumption was noted during entire lactation period measured between lactation days 1 to 4, 4 to 7 and 7 to 13 when compared with the vehicle control group (within the in-house historical control range).

These changes are considered as test item-related due to noted treatment related reduced mean body weight/percent change in body weight gain during pre-mating and gestation periods, reduced food consumption during pre-mating and gestation periods and also due to effects on reproductive/developmental end points at this dose level.

Please also refer to the field "Attached background material" below.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- 450 mg/kg bw/day: microscopically, test item-related changes were observed in prostate (12/12 males) and seminal vesicle (6/12 males) but coagulating glands were within normal limits. The histopathological change observed in prostate and seminal vesicles was atrophy of acinar cell type. It was characterised by multifocal areas of minimal to moderate reduction in acinar size of prostate with reduction or absence of luminal secretions. Similarly, the atrophy in seminal vesicle was characterised by focal area of minimal to mild reduction in vesicle lumen with reduction or absence of secretions and increased epithelial folds.

The incidence of atrophy of prostate and seminal vesicles was noted only in main group males and was considered as test item-related effect. These noted microscopic observations from main group males are not in the in-house historical control findings. This change was found to be reversible at the end of the 14-day recovery period.

Please also refer to the field "Attachd background material" below.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

Main group:
- 450 mg/kg bw/day:
- 6/12 females noted with normal oestrus cycles until sperm positive with the first male during cohabitation period.
- 2/12 females noted with irregular oestrus cycles until sperm positive with the proven male during extended cohabitation period.
- 4/12 females noted with irregular oestrus cycles during initial (14 days with first male) and extended (7 days with proven male) cohabitation periods. These females were not confirmed with positive signal of mating/ sperm positive until 21 days of cohabitation period.

These changes are considered as test item-related due to noted treatment related clinical signs of systemic toxicity in some of the animals, reduced mean body weight, reduced percent change in mean body weight gain, reduced mean food consumption and effects on other reproductive/developmental end points at this dose level. The noted mean oestrus cycle length of the 450 mg/kg bw/day group is within in-house historical control range during pre-mating period. However, a total of 6 females were noted with irregular cycles during mating period until confirmation of mating and or termination of mating from this dose level.

Please also refer to the field "Attached background material" below.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Main group:
1) Males
- 450 mg/kg bw/day: 6/12 males were confirmed with mating during the cohabitation period with a mating index of 50.0%. The statistically significant decrease (p< 0.05) in number of males evidenced with mating when compared with the vehicle control group is considered as test item- related due to noted treatment related reduced mean body weight/percent change in body weight gain, reduced food consumption, effects on reproductive/developmental end points and also due to noted gross/histopathological changes in male accessary sex organs at this dose level.

Furthermore, a total of 6/12 males were confirmed as fertile by impregnating a female or siring a litter with a fertility index of 50.0 %. A statistically significant decrease (p < 0.05) in number of males confirmed as fertile was observed when compared with the vehicle control group. This decrease is considered test item-related due to noted treatment related reduced mean body weight/percent change in body weight gain, reduced food consumption and also due to noted effects on reproductive/developmental end points and due to noted gross/histopathological changes in male accessary sex organs at this dose level.

2) Females
- 450 mg/kg bw/day: 8/12 females were confirmed as sperm positive during vaginal smear examination with a mating index of 66.7 %. A statistically significant decrease (p < 0.05) in number of females confirmed as mated was observed when compared with the vehicle control group. This decrease is considered test item-related due to noted treatment related reduced mean body weight/percent change in body weight gain, reduced food consumption and also due to noted effects on other reproductive/developmental end points at this dose level.

Furthermore, a total of 5/12 mated females were confirmed with presence of implantations / presence of live or dead fetuses / evidence of parturition with a fertility index of 41.7 %. A statistically significant decrease (p < 0.05) in number of females confirmed as fertile was observed when compared with the vehicle control group. This decrease is considered test item-related due to noted treatment related reduced mean body weight/percent change in body weight gain, reduced food consumption and also due to noted effects on other female reproductive/developmental end points at this dose level.

A total of 5/8 mated females were confirmed as pregnant / with evidence of implantation sites with a fecundity index of 62.5%. A statistically significant decrease (p < 0.05) in number of females confirmed with evidence of implantation sites was noted when compared with the vehicle control group. This decrease is considered test item-related due to noted treatment related reduced mean body weight/percent change in body weight gain, reduced food consumption and also due to noted effects on other female reproductive/developmental end points at this dose level.

A total of 4/5 females were confirmed with live born pups with a gestation index of 80.0%. A statistically significant decrease (p < 0.05) in number of females confirmed as littered with live born pups was noted when compared with the vehicle control group. This decrease is considered test item-related due to noted treatment related reduced mean body weight/percent change in body weight gain, reduced food consumption and noted effects on other female reproductive/developmental end points at this dose level.

A mean number of 4.40 post-implantation losses with a percentage of 48.41 % were noted. An increase in number and percentage of post-implantation losses (P < 0.05) was noted when compared with vehicle control group. This increase is considered test item- related due to noted treatment related reduced mean body weight/percent change in body weight gain, reduced food consumption and due to noted effects on female reproductive/developmental end points (mating, fertility, fecundity and gestation indices) at this dose level.

Please also refer to the field "Attached background material" below.

Details on results (P0)

CLINICAL SIGNS
Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no clinical signs of toxicity noted in the daily clinical signs observations in any of the animals of both sexes throughout the experimental period. Furthermore, the detailed clinical examination conducted once in a week did not reveal any changes in any of the animals of both sexes.

Recovery groups:
- 200 mg/kg bw/day: there were no clinical signs of toxicity noted in the daily clinical signs observations in any of the animals of both sexes throughout the experimental period. Furthermore, the detailed clinical examination conducted once in a week did not reveal any changes in any of the animals of both sexes.

MORTALITY
Main groups:
- 50, 100, 200 and 450 mg/kg bw/day: there were no increasead mortality or morbidity noted in any of the animals of both sexes throughout the experimental period.

Recovery groups:
- 200 and 450 mg/kg bw/day: there were no increased mortality or morbidity noted in any of the animals of both sexes throughout the experimental period.

BODY WEIGHT AND WEIGHT CHANGES
1) Males until termination and females during pre-mating and mating period; non-pregnant females with and without positive mating signal during post-mating period

Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 in both sexes throughout the experimental period.

Recovery groups:
- 200 mg/kg bw/day: there were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 in both sexes throughout the experimental period.

2) Gestation period
Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no changes noted in mean gestational body weight and percent change in mean gestational body weight gain at the dose groups when compared with the vehicle control group.

3) Lactation period
Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no changes noted in mean lactation body weight and percent change in mean lactation body weight gain at the tested dose groups when compared with the vehicle control group.

4) Terminal body weight
Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no changes noted in mean terminal body weights at the tested dose groups of both sexes when compared with vehicle control group.

Recovery group:
- 200 mg/kg bw/day: there was no change noted in mean terminal body weight at the tested dose group of both sexes when compared with vehicle control group.

FOOD CONSUMPTION
1) Premating period (males and females) and postmating (males and non-pregnant females with and without positive mating signal)

Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no changes noted in mean food consumption in both sexes during pre-mating and post-mating periods when compared with the vehicle control group.

Recovery groups:
- 200 mg/kg bw/day: there were no changes noted in mean food consumption in both sexes during pre-mating and post-mating periods when compared with the vehicle control group.

2) Gestation period
Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no changes noted in mean food consumption during gestation period at the tested dose groups when compared with the vehicle control group.

3) Lactation period
Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no changes noted in mean food consumption during lactation period at the tested dose groups when compared with the vehicle control group.

OPHTALMOLOGICAL FINDINGS
Main groups:
- 0, 50, 100, 200 and 450 mg/kg bw/day: there were no ocular changes observed in any of the animals of both sexes during ophthalmological examination (pre-treatment and shortly before scheduled sacrifice).

Recovery groups:
- 0, 200 and 450 mg/kg bw/day: there were no ocular changes observed in any of the animals of both sexes during ophthalmological examination (pre-treatment and shortly before scheduled sacrifice)

HAEMATOLOGICAL FINDINGS
Main groups:
- 50, 100, 200 and 450 mg/kg bw/day: there were no test item-related changes noted in mean haematological parameters at all tested main groups of both sexes when compared with the vehicle control group.

Recovery groups:
200 and 450 mg/kg bw/day: there were no test item-related changes noted in mean haematological parameters at all tested recovery groups of both sexes when compared with the vehicle control group.

Main groups and recovery groups:
The following statistically significant changes were noted in haematological parameters when compared with vehicle control groups.
- decrease in mean corpuscular hemoglobin concentrations in main group males of the 50, 200 and 450 mg/kg bw/day dose levels (within the in-house historical control range);
- increase of mean neutrophils (%) in main group males of the 200 mg/kg bw/day dose level (within the in-house historical control range);
- increase in the mean activated prothrombin time in main group females of the 100 mg/kg bw/day dose level (within the in-house historical control range);
- increase in the mean absolute lymphocyte count in recovery group males of the 200 mg/kg bw/day dose level (within the in-house historical control range);
- increase in the mean reticulocyte count (%) and mean prothrombin time in recovery group females of the 450 mg/kg bw/day dose level (within the in-house historical control range).

These changes are considered as incidental and unrelated to treatment as the changes did not occurr in a dose dependent manner and also all the obtained mean values for these significant changes are within in-house historical control range of same species and strain.

CLINICAL BIOCHEMISTRY FINDINGS
Main groups:
- 50, 100, 200 and 450 mg/kg bw/day: there were no test item-related changes noted in mean clinical chemistry parameters at all tested main groups of both sexes when compared with the vehicle control groups.

Recovery groups:
- 200 and 450 mg/kg bw/day: there were no test item-related changes noted in mean clinical chemistry parameters at all tested recovery groups of both sexes when compared with the vehicle control groups.

Main groups and recovery groups:
The following statistically significant changes were noted in clinical chemistry parameters when compared with vehicle control groups.
- increase in mean creatinine levels in main group males of the 50 and 450 mg/kg bw/day dose levels (within the in-house historical control range);
- increase in mean calcium levels in main group of the 50 and 450 mg/kg bw/day dose levels (within the in-house historical control range);
- decrease in mean cholinesterase levels in main group of the 200 mg/kg bw/day dose level (within the in-house historical control range);
- increase in mean alanine aminotransferase in main group females of the 100 mg/kg bw/day dose level (within the in-house historical control range);
- decrease in mean alkaline phosphatase levels in recovery group females of the 450 mg/kg bw/day dose level (within the in-house historical control range).

These changes are considered as incidental and unrelated to treatment as the changes did not occurre in a dose dependent manner and also all the obtained mean values for these significant changes are within in-house historical control range of same species and strain.

URINALYSIS
Main groups:
- 50, 100, 200 and 450 mg/kg bw/day: there were no test item-related changes noted in mean urinalysis parameters at all tested main group males when compared with the vehicle control group.

- 450 mg/kg bw/day: statistically significant decrease in mean urine volume was observed in males (4.5 ± 1.5 mL; p <0.05) when compared with vehicle control group (9.4 ± 2.6 mL). This noted reduction in mean value is below the in-house historical control range. However, the noted reduction was considered as incidental and unrelated to treatment as the rest of the urine physical, chemical and microscopic parameters were unaffected at this dose level.

Recovery groups:
- 200 and 450 mg/kg bw/day: there were no test item-related changes noted in mean urinalysis parameters in all recovery groups of both sexes when compared with the vehicle control group.

BEHAVIOUR (FUNCTIONAL FINDINGS)
Main groups and recovery groups:
- 50, 100, 200 and 450 mg/kg bw/day: the neurological/functional observations did not reveal any changes in any of the animals of both sexes from all the tested dose groups performed towards end of the dosing period for main groups (on day 45 for males and on lactation day 13 for females/dams) and performed towards end of recovery period for recovery groups (day 65).

There were no test item-related changes noted in mean fore/hind limb grip strengths, mean motor activity assessments and mean hind limb foot splay at all tested dose groups of both sexes when compared with vehicle control groups during conduct of neurological/functional examinations.

Main group:
- 100 mg/kg bw/day: the noted statistically significant increase in mean movement counts during motor activity assessment in males when compared with vehicle control group is considered as incidental and toxicologically not relevant and also the obtained mean value is within in-house historical control range.

Recovery groups:
200 and 450 mg/kg bw/day: the noted statistically significant decrease in mean number of grooming in females of the 200 mg/kg bw/day group and statistically significant decrease in mean hind limb grip strength in females of the 450 mg/kg bw/day group when compared with vehicle control group can be considered as incidental as there were no such changes noted in main groups and also no effects noted in forelimb grip strength of same group animals conducted at similar time. Also, the noted mean value for mean hind limb grip strength in the females of the 450 mg/kg bw/day group was within in-house historical control range.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN WEIGHT / BODY WEIGHT RATIOS
Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no test item-related changes noted in mean absolute and relative organ weights at the tested dose groups of both sexes when compared with vehicle control group.

- 50 and 100 mg/kg bw/day: the noted statistically significant increase (p < 0.05) in mean absolute weight of spleen (within the in-house historical control data normal range, but above the acceptable limits) and statistically significant decrease (p < 0.05) in mean absolute and relative weight of ovaries in females (Absolute weight: within the in-house historical control range for 100 mg/kg bw/day group; below the in-house historical control range for 50 mg/kg bw/day group; relative weight: below the in-house historical control range for both groups) when compared with vehicle control group are considered as incidental and unrelated to treatment as there were no gross pathological changes noted in any of these organs during necropsy from any of the animals of these dose groups and there is no dose-response relationship.

- 450 mg/kg bw/day: in males and females, the following statistically significant changes were noted when compared with vehicle control group:
- increase in mean relative weight of adrenals, brain, liver and lungs in main group males (within the in-house historical control range);
- decrease in mean absolute weight of ovaries and thyroid along with parathyroid in main group females (within the in-house historical control range)
- increase in mean relative weight of thymus, spleen, uterus with cervix, heart, brain, liver, lungs, pituitary gland and thyroid along with parathyroid in main group females (within the in-house historical control range, except for spleen and lungs);

Recovery group:
- 200 mg/kg bw/day: there were no test item-related changes noted in mean absolute and relative organ weights of both sexes when compared with vehicle control group.

- 450 mg/kg bw/day: in males and females, the following statistically significant changes were noted when compared with vehicle control group:
- increase in mean relative weight of thymus in recovery group males (within the in-house historical control range);
- increase in mean relative weight of kidneys, brain and liver in recovery group females (within the in-house historical control range)

The observed changes in mean absolute/relative weights of organs such as, spleen, ovaries, kidneys, uterus with cervix, heart, thymus, adrenals, brain, liver, lungs, pituitary gland and thyroid along with parathyroid are considered as incidental in both sexes as there were no gross or histopathological changes noted at this dose level during necropsy or microscopic examinations and also the obtained values are within in-house historical control ranges.

Please also refer to the field "Attached background material" below.

HISTOPATHOLOGICAL FINDINGS - NON-NEOPLASTIC
- 0 and 450 mg/kg bw/day: a few other microscopic findings were observed in the study such as ultimobranchial cyst in thyroid, cystic tubule and infiltration of mononuclear cells in interstitium in kidneys and accessory cortical tissue in adrenals. However, these findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats of this age (Elizabeth McInnes, 2012)*.

GROSS PATHOLOGICAL FINDINGS
Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no gross pathological changes noted at the dose groups during conduct of necropsy.

Recovery group:
- 200 mg/kg bw/day: there were no gross pathological changes noted at the dose group during conduct of necropsy.

REPRODUCTIVE FUNCTION: OESTROUS CYCLE
Main groups:
- 50, 100, 200 and 450 mg/kg bw/day: there were no irregularities observed in the oestrus cyclicity of all main group females during pre-mating period. The mean length of oestrus cycle (days) per dam during pre-mating period was unaffected at all dose groups when compared with vehicle control group.

REPRODUCTION PERFORMANCE
Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no changes observed in total litter size at the tested dose groups when compared with the vehicle control group.

- 0, 50, 100 and 200 mg/kg bw/day: a total of 12/12, 12/12, 12/12, and 12/12 males were confirmed with mating during the cohabitation period with a mating index of 100.0%, 100.0%, 100.0%, and 100.0% for 0, 50, 100 and 200 mg/kg bw/day, respectively. Furthermore, a total of 11/12, 11/12, 11/12, and 10/12 males were confirmed as fertile by impregnating a female or siring a litter with a fertility index of 91.7 %, 91.7 %, 91.7 % and 83.3 % for 0, 50, 100 and 200 mg/kg bw/day, respectively. There were no statistically significant differences noted for male fertility index at the tested dose groups when compared with vehicle control group.

- 0, 50, 100 and 200 mg/kg bw/day: a total of 12/12, 12/12, 12/12, and 12/12 females were confirmed as sperm positive during vaginal smear examination with a mating index of 100.0%, 100.0%, 100.0%, 66.7% and 100.0% for 0, 50, 100 and 200 mg/kg bw/day, respectively. Furthermore, a total of 11/12, 11/12, 11/12, and 10/12 mated females were confirmed with presence of implantations / presence of live or dead fetuses / evidence of parturition with a fertility index of 91.7 %, 91.7 %, 91.7 %, and 83.3 % for 0, 50, 100 and 200 mg/kg bw/day, respectively. There were no statistically significant differences noted for female fertility indices at the tested dose groups when compared with the vehicle control group.

- 0, 50, 100, 200 and 450 mg/kg bw/day: a total of 12 pairs were left for cohabitation initially from each group. The mean pre-coital interval was 10.50, 6.83, 8.67, 7.17 and 11.38 days for 0, 50, 100, 200 and 450 mg/kg bw/day, respectively. There were no statistically significant differences noted for this parameter at any of the tested dose groups when compared with the vehicle control group.

- 0, 50, 100, 200 and 450 mg/kg bw/day: the mean gestation length [day of confirmed as successfully mated to day of parturition] was 22.36, 22.18, 22.18, 22.60 and 22.40 days for 0, 50, 100, 200 and 450 mg/kg bw/day, respectively. There were no statistically significant differences noted for this parameter at any of the tested dose groups when compared with the vehicle control group.

- 0, 50, 100 and 200 mg/kg bw/day: a total of 11/12, 11/12, 11/12 and 10/12 mated females were confirmed as pregnant / with evidence of implantation sites with a fecundity index of 91.7 %, 91.7 %, 91.7 % and 83.3 % for 0, 50, 100 and 200 mg/kg bw/day, respectively. There were no statistically significant differences noted for this parameter at the tested dose groups when compared with the vehicle control group.

- 0, 50, 100 and 200 mg/kg bw/day: a total of 11/11, 11/11, 11/11and 10/10 females were confirmed with live born pups with a gestation index of 100.0 %, 100.0 %, 100 % and 100.0 %, for 0, 50, 100 and 200 mg/kg bw/day, respectively.

- 0, 50, 100 and 200 mg/kg bw/day: a mean number of 0.82, 0.36, 0.36, and 0.60 post-implantation losses with a percentage of 6.67 %, 3.39 %, 3.39 %, and 5.30 % were noted for 0, 50, 100 and 200 mg/kg bw/day, respectively. There were no statistically significant changes noted for this end point at tested dose groups when compared with the vehicle control group.

ENDOCRINE FINDINGS
Main groups and recovery groups:
- 50, 100, 200 and 450 mg/kg bw/day: there were no test item-related changes noted in serum T3, T4 and TSH levels at any of the tested main and recovery groups from both sexes when compared with vehicle control groups.

- 50, 200 and 450 mg/kg bw/day: the statistically significant increase in mean serum T3 levels in females of the 50 mg/kg bw/day dose level (main group) and statistically significant increase in mean serum TSH levels in females of the 200 mg/kg bw/day dose level (main group) are considered as incidental and unrelated to treatment as the changes are not dose dependent and also the obtained values are within in-house historical control range of same species and strain.

Please also refer to the field "Attached background material" below.

*Reference:
- Elizabeth F. McInnes, 2012, Background lesions in Laboratory animals: A color Atlas, Saunders, Elsevier, Edinburgh.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

(reproductive toxicity)

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive performance

2

Key result

Dose descriptor:

NOAEL

Remarks:

(general toxicity)

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Main group:
- 450 mg/kg bw/day: decrease in mean number of live pups born, mean live birth index (%), mean number of survived pups and pup survival index (%) during lactation period and increase in mean number of dead pups born were noted when compared with the vehicle control group. These changes are statistically significant for mean number of female live pups born (below the in-house historical control range), mean number of females and combined sex survived pups on lactation day 4 (below the control range based of in-house historical control data for pup survival index during lactation days 1 to 4), mean number of survived pups for combined sex on lactation day 7 and 13 (below the control range based on in-house historical control data for pup survival index during lactation day 5 to 7 and 8 to 13), mean live birth index (below the in-house historical control range) and mean pup survival index on lactation day 4, 7 and 13 (below the in-house historical control range).

The occurred changes are considered as test item-related due to noted treatment related reduced mean body weight/percent change in body weight gain during
pre-mating/gestation/lactation periods and also due to noted effects on other reproductive/developmental end points at this dose level.

- 450 mg/kg bw/day: the mean number of postnatal loss per litter was noted as 0.40 with a percentage of 22.00% (above the in-house historical control range) and these values are statistically significant (P < 0.05) when compared with vehicle control group. These effects are considered test item-related due to noted reduced mean body weight/percent change in body weight gain, reduced food consumption, increased pup mortalities, reduced pup survival index and reduced pup weights at this dose level.

Please also refer to the field "Attached background material" below.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 450 mg/kg bw/day: a decrease in mean live pup weight of both sexes was noted and this change is statistically significant for mean female pup weight when compared with vehicle control group only on postnatal day 1 (within the in-house historical control range). Pup weights were only slightly lower than control group data thereafter until postnatal day 13. This effect can be considered as a secondary effect due to noted test item related maternal effects at this dose level.

Please also refer to the field "Attached background material" below.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

CLINICAL SIGNS
Main groups:
- 50, 100; 200 and 450 mg/kg bw/day: there were no external abnormalities or behavioural changes noted for any of the pups during daily observation from all dose group litters during postnatal period. All pups were noted with normal behaviour during daily observations.

MORTALITIY / VIABILITY
Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no changes observed in birth parameters such as number of live pups born and live birth index at the tested dose groups when compared with the vehicle control group. Also, the pup survival index per litter during lactation period was unaffected by the test item at tested dose groups when compared with the vehicle control group.

- 50, 100 and 200 mg/kg bw/day: there were no incidences of postnatal loss at the tested dose groups and vehicle control group during lactation period.

BODY WEIGHTS AND WEIGHT CHANGES
Main groups:
- 50, 100 and 200 mg/kg bw/day: there were no test item-related changes noted in mean pup (both male and female) weight per litter recorded on postnatal day 1, 4, 7 and 13 at tested dose group when compared with vehicle control group litters. The noted statistically significant increase in mean female pup weight in the 200 mg/kg bw/day group on postnatal day 13 is considered as incidental and toxicologically not relevant and also within the in-house historical control range.

ANOGENITAL DISTANCE
Main groups:
- 50, 100, 200 and 450 mg/kg bw/day: there were no changes noted in mean pup (both male and female) anogenital distance measurement and ratio per litter recorded on postnatal day 4 for all dose groups when compared with vehicle control group.

NIPPLE RETENTION IN MALE PUPS
Main groups:
- 0, 50, 100, 200 and 450 mg/kg bw/day: there were no occurrences of nipples in any of the male pups examined on the postnatal day 13 from any of the tested dose groups and vehicle control group litters.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN/BODY WEIGHT RATIOS
Main groups:
- 50, 100, 200 and 450 mg/kg bw/day: there were no changes noted in mean absolute and relative thyroid weights collected from postnatal day 13 pups at all dose groups of both sexes when compared with vehicle control group.

GROSS PATHOLOGICAL FINDINGS
There were no test item –related gross pathological changes observed during scheduled necropsy in all pups and in those pups which died at birth or during lactation period . Single incidence of unilateral short limb was observed during external gross pathological evaluation in a male pup died at birth belonging to high dose group. This finding was considered as an isolated incidental finding without any relation to administration of test item as it lacks dose correlation.

HISTOPATHOLOGICAL FINDINGS
- 0 and 450 mg/kg bw/day: there were no histopathological changes noted in any of the postnatal day 13 pups examinated from vehicle control and high dose groups, except a single incidental finding, i.e. ultimobranchial cyst in thyroid gland from a female pup of vehicle control group.

ENDOCRINE FINDGINGS
Main groups:
- 50, 100, 200 and 450 mg/kg bw/day: there were no test item related changes noted in serum T3, T4 and TSH hormone levels of postnatal day 4 and 13 pups at any of the tested dose group litters when compared with vehicle control group litters.

- 50, 200 and 450 mg/kg bw/day: the statistically significant decrease in serum T4 levels of postnatal day 13 pups at 50 and 450 mg/kg bw/day dose levels and statistically significant decrease in serum TSH levels of postnatal day 4 pups at 200 mg/kg bw/day dose level when compared with vehicle control group are considered as incidental and unrelated to treatment as the changes are not dose dependent and the obtained values are within in-house historical control range of same species and strain.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

(developmental toxicity)

Generation:

F1

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a current combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, barium 4-dodecylphenolate was administered via gavage to groups of male and female Sprague-Dawley rats (n = 12 animals/sex/group) at dose levels of 50, 100, 200 and 450 mg/kg bw/day. The test item was administered once daily for sevens day during premating period, mating period, pregnancy and lactation. A vehicle (corn oil) control group was run concurrently. In addition, three recovery groups with 5 males and 5 females each (0, 200 and 450 mg/kg bw/day) were also run concurrently to determine the reversibility of the test item related effects.

During the observations of the parental male and female rats, no test item-related effects were observed for mortality, opthalmology, haematology, clinical chemistry including serum thyroid hormone levels (T3, T4 and TSH), urinalysis, or neurobehaviour.

At the 450 mg/kg bw/day dose level, some male and female animals were noted with test item-related clinical signs such as perinasal staining, rough coat, lethargy, wet perineum. Animals of the recovery group fully recovered from these clinical signs and were found normal during the recovery period. Furthermore, animals of the 450 mg/kg bw/day dose group were noted with test-item related reduction in body weight and body weight gain as well as in food consumption. Test item-related reduced absoute/relative weights of testes, epididymis, prostate and seminal vesicles with coagulating glands were also noted at this high dose level. Test item-related gross pathological changes in form of small sized prostate and seminal vesicle with coagulating gland and microscopically, test item-related acinar atrophy in the prostate and seminal vesicles were noted in males at the 450 mg/kg bw/day dose level. The recovery group animals of the 450 mg/kg bw/day dose group recovered during the recovery period and were found normal at termination.

At the 450 mg/kg bw/day dose level, some females had an irregular oestrus cycle test item-related reduction in mating and fertility indices were noted in both sexes. The pregnancy and gestation indices were also reduced at this dose level and evidenced as test item-related. The increased post-implantation and postnatal losses at this dose level was also evidenced as test item-related. Test item-related reduced litter size, reduced live birth index per litter, increased number of dead pups at birth and reduced pup survival index were also noted.

The investigation of the pups of the 450 mg/kg bw/day dose level showed test item-related reduced mean pup weight per litter in either sex. There were no occurrences of nipples in male pups examined on postnatal day 13 from all litters and the anogenital distance was also not affected by the test item. No gross pathological changes were noted in any of the survived pups. There were no changes noted in serum thyroid hormone levels (T3, T4 and TSH) measured in postnatal day 4 and 13 pups. There were no changes in mean absolute/relative pup thyroid weights collected on postnatal day 13 and also no histopathological changes of the thyroid.

Based on the test item related clinical signs, and adverse effects on body weight, food consumption, organ weights, gross pathology and histopathology at the 450 mg/kg bw/day dose level, the no observed adverse effect level (NOAEL) for general toxicity is considered to be 200 mg/kg bw/day for males and females. Furthermore, based on test item related effects on reproductive function (irregular oestrus cycle) and reproductive performance (reduced mating index, fertility index, fecundity index and increased post-implantation loss), the NOAEL for reproduction toxicity is considered to be 200 mg/kg bw/day. A NOAEL for developmental toxicity was determined to be 200 mg/kg bw/day based on test item-related effects on live births, pup survival and pup body weight observed at the 450 mg/kg bw/day dose level.